High performance liquid chromatographic determination of the stabilized cyclophosphamide metabolite 4-hydroxycyclophosphamide in plasma and red blood cells

A reversed-phase high performance liquid chromatographic (HPLC) method for the determination of the activated cyclophosphamide (CP) metabolite 4- hydroxycyclophosphamide (4-OHCP) in human plasma and red blood cells is described. 4-OHCP is very unstable in biological matrices. Therefore, it was treated, immediately after sampling, with semicarbazide to form a stable semicarbazone derivative, which was identified with electron spray mass spectrometry. The derivative was analysed with HPLC with ultraviolet (UV) detection at 230 nm. Sample pretreatment consisted of a liquid-liquid extraction with ethyl acetate, the chromatography system was a 25 cm C8 column (particle size 5 μm) with a mobile phase of acetonitrile-0.025 M potassium phosphate buffer (15:85 v/v) pH 6.0. After assay optimisation, the method was validated and stability studies were carried out. The method proved linear in clinically relevant concentrations (50-5000 ng/mL). The lower limit of quantitation was 50 ng/mL. Accuracy and precision were below 7% over the full concentration range. A calibration curve in plasma can also be used for the quantification of 4-OHCP in red blood cells. The derivative was found to be stable for at least 11 months when stored at -70 °C. A pharmacokinetic pilot study in a patient treated with 1000 mg/m² CP in combination with thio TEPA and carboplatin demonstrated the applicability of the assay for the determination of 4-OHCP in plasma and red blood cells.